Non-specific staining is a major problem in immunological determinations, which measure the presence of an antigen on cells or in solutions or which isolate specific cells or proteins on the basis of binding of detection reagents that specifically recognize these antigens. The specific binding of a detection reagent to its target antigen cannot be distinguished from non-specific binding to other structures that are unrelated to the antigen of interest. Ideally, detection reagents used in immunological determinations specifically recognize the antigens of interest, but do not bind to other unrelated molecules on the same test cells or in the same test solution. Monoclonal antibodies raised against the antigen of interest are used most often, but not exclusively, as detection reagents. Polyclonal antibodies or non-immunoglobulin reagents, e.g., cytokines or synthetic antibody-like molecules are used as well, but less frequently.
A major, but not the only, source of non-specific signals in immunological determinations is the interaction of antibodies with Fc-receptors (FcR) on the surface of various blood cells or present in soluble form in blood serum and plasma. Examples of FcR expressing cells are monocytes/macrophages, NK cells, B cells, and platelets. Examples of Fc Receptors that can bind antibodies or antibody complexes are CD16, CD23, CD32, and CD69. Antibody/FcR interactions are mediated through the constant or Fc part of the molecule. The Fc part is also involved in other non-specific antibody interactions which are not mediated through Fc receptors. Removal of the Fc part of antibodies by chemical or enzymatic protein digestion can effectively remove FcR mediated or other non-specific interactions. Disadvantages of these approaches are that the chemical or enzymatic procedures that remove the Fc part of antibodies can damage the antibody molecule and decrease its stability and performance. Digestion conditions need to be carefully optimized for each individual antibody preparation to achieve minimal damage and maximal recovery. This increases the amount of labour and cost involved. The removal of the Fc region may also reduce detection sensitivity. The reason is that the smaller size of the digested antibodies reduces the amount of fluorochrome, enzyme or other label that can be effectively attached to the detection antibody without inhibiting or eliminating its antigen binding ability.
Desirable approaches to prevent non-specific binding of detection antibodies in immunological determinations prevent FcR and other non-specific interactions by non-modified, intact detection antibodies and can be applied generically without need for optimization for different applications and assay formats.